1. Field of the Invention
The present invention relates to a method of removing a gene and method of obtaining the gene by partial homologous recombination of a DNA chain.
2. Description of the Related Art
A DNA library, in particular, a cDNA library, is an extremely useful tool for cloning a gene. Numerous genes have been so far cloned from a cDNA library. A cloned gene is used for determining not only the sequences of the gene but also the amino acid sequence of a protein encoded by the gene, and also used for producing the protein in bacterial and yeast cells in a large amount.
However, cDNAs easily cloned from a cDNA library are limited to those prepared from mRNA (used as a template) expressing in a large amount in cells. Since numerous genes have been now cloned, almost all of the easy-to-clone cDNAs have been cloned. Therefore, it has become difficult to efficiently clone a novel cDNA.
To efficiently clone a novel cDNA from a cDNA library, it is necessary to remove cDNAs already cloned from the cDNA library. To attain this purpose, the following techniques have been established.
As a basic method for removing a cDNA, subtractive hybridization is known.
In the method, mRNAs are prepared from both cells (or tissues) expressing and not expressing a desired gene. Then, cDNAs are synthesized from one of the mRNAs and hybridized with the other mRNAs; with the result that the common cDNAs in both cells are exclusively removed. In this way, a gene expressed specifically in certain tissues and cells can be condensed and isolated.
As the subtractive hybridization, subtractive hybridization performed on membrane and subtractive hybridization using a hydroxyapatite column are known (Hedrick S M et al., Isolation of cDNA clones encoding T cell-specific membrane-associated proteins, “Nature”, (UK), 1984, Vol. 5955, p. 149–53; and Bonaldo MF et al., Normalization and subtraction: two approaches to facilitate gene discovery, “Genome Res”, (USA), 1966, Vol. 9, p 791–806).
However, the on-membrane subtractive hybridization has a problem in that it is difficult to treat many colonies at a time and therefore it is not suitable for reconstructing the entire library. In addition, false positive or false negative error signals are frequently observed. The analysis takes a long time.
On the other hand, the subtractive hybridization using a hydroxyapatite column has a problem. In the case of a cDNA library containing relative long sequences exceeding 3 kb, the possibility of nonspecific hybridization becomes high. Therefore, this subtractive hybridization has been applied only to cDNA library constructed by relatively short cDNAs within about 0.4 to 2.5 kb. A long sequence has a high possibility of containing functionally important genes that encode multifunctional proteins and complex structure proteins. Therefore, the feature of this method which is not applicable to a library containing long sequences is a serious disadvantage of this method. This method has another disadvantage: even though cDNAs are short, the cDNAs derived from the same gene and have the same 3′ terminal and 5′ terminal, but differ in the middle sequence, cannot be distinguished from each other by this method.
Besides these, there is another method frequently used to achieve the same purpose, a differential display method is known (Liang P et al., Analysis of altered gene expression by differential display, “Method Enzymol”, (USA), 1995, vol. 254, p. 304–21). Furthermore, subtractive hybridization method using PCR, which improves the differential display method is also known (Diatchenko L et al., Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes, “Methods Enzymol”, (USA), 1999, Vol. 303, p. 349–80).
However, these methods mentioned above have disadvantages: unless the expression levels of genes significantly differ, electrophoretic patterns show no difference. In addition, false positive and false negative error signals are frequently observed. Furthermore, since clones are not directly obtained, they must be cloned by any means based on PCR products thereof.
On the other hand, as a conventional technique for obtaining a desired gene from a gene library, the on-membrane hybridization is known (Hedrick S M et al., Isolation of cDNA clones encoding T cell-specific membrane-associated proteins, “Nature”, (UK), 1984, Vol. 5955, p. 149–53).
In this method, colonies or plaques appearing on a plate where Escherichia coli (used as a host) are grown are transferred onto a membrane filter. A detection probe is then hybridized to the membrane filter. It is presumed that the colonies or plaques expressing signals may contain a desired gene fragment. Therefore, the colonies or plaque are isolated and cultured, thereby obtaining the desired gene fragment.
However, in this technique, it is not suitable for obtaining a rare gene, since it is difficult to treat a large number of colonies at a time. Furthermore, false positive and false negative error signals are frequently observed, so that analysis requires a long time.
Furthermore, a method using liquid phase hybridization is known (Invitrogen Instruction Manual, Gene Trapper cDNA Positive Selection System, Cat. No. 10356-020).
In this method, a library is constructed by using a vector having an M13 replication origin. This is converted into a library containing circular single-stranded gene fragments within Escherichia coli (E. coli) or in vitro. The obtained library is then subjected to hybridization with a labeled probe in a liquid phase, and then, hybridized DNA is isolated by binding the label onto a solid phase via a substance recognizing the label. After it is recovered from the solid phase, the hybridized DNA is converted into a double stranded DNA, which is then introduced into E. coli to transform it. In this manner, a desired gene can be obtained.
However, this method has a problem: when no less than 70% of sequences of DNA are homologous to other DNA, the probes non-specifically hybridize to homologous DNA. Therefore, the probe cannot be designed so as to bind to an desired portion.
For other methods to obtain a desired gene from a library, a cloning method using a RecA protein is disclosed (National Patent Publication No. 6-500926).
In this method, first, a triple-stranded DNA is formed with a labeled probe in a liquid phase, and then isolated by binding it onto a solid phase via a substance specifically recognizing the label. The DNA is recovered from the solid phase and thereafter introduced into E. coli to transform it. In this manner, a desired gene can be obtained.
However, this method also has a problem. A circular double stranded DNA is only used in the reaction. Therefore, the specificity and efficiency are not always high.
Furthermore, cloning in which a DNA extension reaction is inhibited by a triple-stranded structure is disclosed (Japanese Patent Application KOKAI publication No. 11-206381).
In this method, a library gene is cleaved with a restriction enzyme and a triple-stranded structure is formed at a cleaved site. A triple-stranded structure will be formed in a clone containing a desired gene fragment. Consequently, the clone having a triple-stranded structure formed therein will no longer used as a substrate for a DNA polymerase extension reaction. Therefore, other clones will be only extended. After the reaction, the triple-stranded chain is dissociated and DNA is re-annealed. In this manner, a desired clone can be obtained.
However, this method has a problem: as a degree of specificity increases, the efficiency is decreased. In other words, as the efficiency increases, a degree of specificity decreases.
The present inventors have already disclosed a method of constructing a DNA library having an increased content of a desired gene by using a RecA protein; however, this method fails to use a linear DNA library and thus a further improvement has been desired (Japanese Patent Application KOKAI publication No. 2001-346576).